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The role of microglia and the TLR4 pathway in neuronal apoptosis and vasospasm after subarachnoid hemorrhage.

Hanafy KA - J Neuroinflammation (2013)

Bottom Line: Our results suggest that SAH pathology could have different phases.These results could explain why therapies tailored to aSAH patients have failed for the most part.Perhaps a novel strategy utilizing immunotherapies that target Toll like receptor signaling and microglia at different points in the patient's hospital course could improve outcomes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of NeuroCritical Care, Department of Neurology, Harvard Medical School, Beth Israel Deaconess Medical Center, The Center for Life Science, 3 Blackfan Circle, Boston, MA 02215, USA. khanafy@bidmc.harvard.edu

ABSTRACT

Background: Although microglia and the Toll-like receptor (TLR) pathway have long been thought to play a role in the pathogenesis of aneurysmal subarachnoid hemorrhage (aSAH), thus far only correlations have been made. In this study, we attempted to solidify the relationship between microglia and the TLR pathway using depletion and genetic knockouts, respectively.

Methods: Subarachnoid hemorrhage was induced in TLR4-/-, TRIF-/-, MyD88-/- and wild type C57BL/6 mice by injecting 60 μl of autologous blood near the mesencephalon; animals were euthanized 1 to 15 days after SAH for immunohistochemical analysis to detect microglia or apoptotic cells. Lastly, microglial depletion was performed by intracerebroventricular injection of clodronate liposomes.

Results: On post operative day (POD) 7 (early phase SAH), neuronal apoptosis was largely TLR4-MyD88-dependent and microglial-dependent. By POD 15 (late phase SAH), neuronal apoptosis was characterized by TLR4- toll receptor associated activator of interferon (TRIF)-dependence and microglial-independence. Similarly, vasospasm was also characterized by an early and late phase with MyD88 and TRIF dependence, respectively. Lastly, microglia seem to be both necessary and sufficient to cause vasospasm in both the early and late phases of SAH in our model.

Conclusion: Our results suggest that SAH pathology could have different phases. These results could explain why therapies tailored to aSAH patients have failed for the most part. Perhaps a novel strategy utilizing immunotherapies that target Toll like receptor signaling and microglia at different points in the patient's hospital course could improve outcomes.

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Neuronal apoptosis time course in subarachnoid hemorrhage model. A. Merge of DAPI and TUNEL staining in sham and subarachnoid hemorrhage (SAH) along entire time course from post-operative days (PODs) 1 to 15. Scale Bar: 10 μm. B. Nissel stain from atlas.brain-map.org showing specific area of dentate gyrus that was used for quantification of neuronal apoptosis. C. Apoptotic neurons quantified in dentate gyrus on each POD. Student’s t-test for each POD showed significance between sham and SAH on POD 5 (P <0.02), 7 (P <0.0001), 10 (P <0.01) and 15 (P <0.0001). Error bars are standard deviations and N = 4 per group.
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Figure 2: Neuronal apoptosis time course in subarachnoid hemorrhage model. A. Merge of DAPI and TUNEL staining in sham and subarachnoid hemorrhage (SAH) along entire time course from post-operative days (PODs) 1 to 15. Scale Bar: 10 μm. B. Nissel stain from atlas.brain-map.org showing specific area of dentate gyrus that was used for quantification of neuronal apoptosis. C. Apoptotic neurons quantified in dentate gyrus on each POD. Student’s t-test for each POD showed significance between sham and SAH on POD 5 (P <0.02), 7 (P <0.0001), 10 (P <0.01) and 15 (P <0.0001). Error bars are standard deviations and N = 4 per group.

Mentions: To determine the optimal time to visualize vasospasm, we performed a time course of subarachnoid hemorrhage from PODs 1 through 15 (Figure 1). The dimensions of the middle cerebral artery were analyzed at the level of the hippocampus for consistency, as shown in Figure 1C. We found that the difference between sham and wild type (WT) SAH vasospasm was maximal on days 3 and 10. There was no difference between sham and WT SAH on POD 5, indicating resolution of vasospasm by Day 5, followed by delayed vasospasm beginning on Day 7 and plateauing on Day 10 (Figure 1). To determine if neuronal cell death correlated with vasospasm in our model, we quantified TUNEL positive cells from days 1 through 15 in the dentate gyrus of the hippocampus. Similarly, cell death also had a bimodal distribution, peaking on PODs 7 and 15 (Figure 2).


The role of microglia and the TLR4 pathway in neuronal apoptosis and vasospasm after subarachnoid hemorrhage.

Hanafy KA - J Neuroinflammation (2013)

Neuronal apoptosis time course in subarachnoid hemorrhage model. A. Merge of DAPI and TUNEL staining in sham and subarachnoid hemorrhage (SAH) along entire time course from post-operative days (PODs) 1 to 15. Scale Bar: 10 μm. B. Nissel stain from atlas.brain-map.org showing specific area of dentate gyrus that was used for quantification of neuronal apoptosis. C. Apoptotic neurons quantified in dentate gyrus on each POD. Student’s t-test for each POD showed significance between sham and SAH on POD 5 (P <0.02), 7 (P <0.0001), 10 (P <0.01) and 15 (P <0.0001). Error bars are standard deviations and N = 4 per group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750560&req=5

Figure 2: Neuronal apoptosis time course in subarachnoid hemorrhage model. A. Merge of DAPI and TUNEL staining in sham and subarachnoid hemorrhage (SAH) along entire time course from post-operative days (PODs) 1 to 15. Scale Bar: 10 μm. B. Nissel stain from atlas.brain-map.org showing specific area of dentate gyrus that was used for quantification of neuronal apoptosis. C. Apoptotic neurons quantified in dentate gyrus on each POD. Student’s t-test for each POD showed significance between sham and SAH on POD 5 (P <0.02), 7 (P <0.0001), 10 (P <0.01) and 15 (P <0.0001). Error bars are standard deviations and N = 4 per group.
Mentions: To determine the optimal time to visualize vasospasm, we performed a time course of subarachnoid hemorrhage from PODs 1 through 15 (Figure 1). The dimensions of the middle cerebral artery were analyzed at the level of the hippocampus for consistency, as shown in Figure 1C. We found that the difference between sham and wild type (WT) SAH vasospasm was maximal on days 3 and 10. There was no difference between sham and WT SAH on POD 5, indicating resolution of vasospasm by Day 5, followed by delayed vasospasm beginning on Day 7 and plateauing on Day 10 (Figure 1). To determine if neuronal cell death correlated with vasospasm in our model, we quantified TUNEL positive cells from days 1 through 15 in the dentate gyrus of the hippocampus. Similarly, cell death also had a bimodal distribution, peaking on PODs 7 and 15 (Figure 2).

Bottom Line: Our results suggest that SAH pathology could have different phases.These results could explain why therapies tailored to aSAH patients have failed for the most part.Perhaps a novel strategy utilizing immunotherapies that target Toll like receptor signaling and microglia at different points in the patient's hospital course could improve outcomes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of NeuroCritical Care, Department of Neurology, Harvard Medical School, Beth Israel Deaconess Medical Center, The Center for Life Science, 3 Blackfan Circle, Boston, MA 02215, USA. khanafy@bidmc.harvard.edu

ABSTRACT

Background: Although microglia and the Toll-like receptor (TLR) pathway have long been thought to play a role in the pathogenesis of aneurysmal subarachnoid hemorrhage (aSAH), thus far only correlations have been made. In this study, we attempted to solidify the relationship between microglia and the TLR pathway using depletion and genetic knockouts, respectively.

Methods: Subarachnoid hemorrhage was induced in TLR4-/-, TRIF-/-, MyD88-/- and wild type C57BL/6 mice by injecting 60 μl of autologous blood near the mesencephalon; animals were euthanized 1 to 15 days after SAH for immunohistochemical analysis to detect microglia or apoptotic cells. Lastly, microglial depletion was performed by intracerebroventricular injection of clodronate liposomes.

Results: On post operative day (POD) 7 (early phase SAH), neuronal apoptosis was largely TLR4-MyD88-dependent and microglial-dependent. By POD 15 (late phase SAH), neuronal apoptosis was characterized by TLR4- toll receptor associated activator of interferon (TRIF)-dependence and microglial-independence. Similarly, vasospasm was also characterized by an early and late phase with MyD88 and TRIF dependence, respectively. Lastly, microglia seem to be both necessary and sufficient to cause vasospasm in both the early and late phases of SAH in our model.

Conclusion: Our results suggest that SAH pathology could have different phases. These results could explain why therapies tailored to aSAH patients have failed for the most part. Perhaps a novel strategy utilizing immunotherapies that target Toll like receptor signaling and microglia at different points in the patient's hospital course could improve outcomes.

Show MeSH
Related in: MedlinePlus